Phosphatidylinositol 3,4,5-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

Abstract

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins.

Cellular and molecular interactions of phosphoinositides and peripheral proteins

“…One such approach was recently developed for proteins expressed with a GFP tag (Kim et al 2013). Synthetic PIPs have also been useful for rapid screening of PIP-binding (Rowland et al 2012) and cellular identification of PIP-binding proteins using PIP-activity probes and proteomics (Rowland et al 2011). In fact, this is an emerging area of research and new assays to globally investigate PIP-binding proteins are nicely reviewed by Michael Best in this special issue (Best 2013).…”

“…Furthermore, proteomics and lipidomics has increased our awareness of the large number of PIP-binding proteins in nature (van Meer 2005; Clark et al 2011; Rowland et al 2011; Best 2013; Catimel et al 2013; Oxley et al 2013) some of which have potential therapeutic value or unique PIP-binding properties. The future research on PIP-binding interactions will encompass more interdisciplinary studies of both computational predictions and validations of these conjectures.…”

Turn-ON fluorescent affinity labeling using a small bifunctional O-nitrobenzoxadiazole unit

“…For this reason, small alkyne tags that are suitable for later conjugation with azide-uorescent or azide-biotin compounds are widely used in in vitro experiments, despite the additional conjugation step. 3,4,6 Otherwise, the design and synthesis of complex multifunctional probes is generally carried out by trial and error, in the absence of information regarding the structure-activity relationship (SAR). To overcome this bottleneck, we have designed a simple chemical probe bearing a small bifunctional O-NBD unit (Fig.…”

Turning the spotlight on protein–lipid interactions in cells

“…These bifunctional probes were able to bind with known PA or PIPns receptors even though bulky benzophenones and linkers bearing alkynes were introduced into the lipid headgroups [36,38••]. Notably, one of the bifunctional probes derived from PI(3,4,5)P 3 ( 3 ) has been utilized for a proteomic photocrosslinking study in cell extracts [38••] (Figure 2a).…”

“…These bifunctional probes were able to bind with known PA or PIPns receptors even though bulky benzophenones and linkers bearing alkynes were introduced into the lipid headgroups [36,38••]. Notably, one of the bifunctional probes derived from PI(3,4,5)P 3 ( 3 ) has been utilized for a proteomic photocrosslinking study in cell extracts [38••] (Figure 2a). The photolabeled proteins were detected by in-gel fluorescence after a click reaction with rhodamine and identified by mass spectrometry after biotinylation and affinity chromatography, yielding both known and new PI(3,4,5)P3-binding proteins with a total number of 265.…”